Unusual apoptotic signaling pathways in cancer cells induced by cephalostatin

(1)

Dissertation zur Erlangung des Doktorgrades der Fakultät für Chemie und Pharmazie der Ludwig-Maximilians-Universität München

Unusual apoptotic signaling pathways in cancer cells

induced by cephalostatin

Anita Rudy aus Altötting

(2)

Erklärung

Diese Dissertation wurde im Sinne von § 13 Abs. 3 bzw. 4 der Promotionsordnung vom 29. Januar 1998 von Frau Prof. Dr. A. M. Vollmar betreut.

Ehrenwörtliche Versicherung

Diese Dissertation wurde selbständig, ohne unerlaubte Hilfe erarbeitet.

München, am 22.02.2007

____________________ Anita Rudy

Dissertation eingereicht am: 22.02.2007

1. Gutachter: Frau Prof. Dr. A. M. Vollmar 2. Gutachter: Herr PD. Dr. C. Culmsee Mündliche Prüfung am: 27.03.2007

(3)

INDEX OF FIGURES

Figure I.1 Fingerprint of cytotoxic profile of cephalostatin 1 and 2 evaluated by the NCI-60 screen (September 2005)...3

Figure I.2 Chemical structure of cephalostatin 1, 2, 10 and 12 ...4

Figure I.3 Overview of apoptotic and necrotic cell death...6

Figure I.4 Classification of caspases based on their prodomain structure or function ...9

Figure I.5 Schematic representation of proteolytic caspase activation (modified from 31) ....10

Figure I.6 Schematic representation of the inhibitor of apoptosis protein family (36)...12

Figure I.7 The extrinsic apoptotic pathway...14

Figure I.8 The intrinsic apoptotic pathway...16

Figure I.9 Simplified illustration of removal of IAP-mediated caspase-inhibition by Smac ...17

Figure I.10 Bcl-2 family members (modified from 40) ...18

Figure I.11 The PIDDosome...19

Figure I.12 Consequences of persistent STAT3 activity. ...21

Figure II.1 flow chamber of a flow cytometer (from 67) ...26

Figure II.2 Histogram of untreated PI-stainded cells ...28

Figure II.3 short interfering RNA (modified from 71). ...40

Figure III.1 Cephalostatin 2 is the most active among the tested cephalostatins...44

Figure III.2 Apoptosis induced by cephalostatin 2, 10 and 12 is dose dependent ...45

Figure III.3 Cephalostatin 2 induced apoptosis is dependent on time...46

Figure III.4 Cephalostatin induces apoptosis in HeLa cells...47

Figure III.5 Morphological alterations in cephalostatin-treated MCF-7 cells...48

Figure III.6 Cephalostatin induces apoptosis in MCF-7 ± caspase-3 cells...49

Figure III.7 Cephalostatin induces concentration dependent apoptosis in SK-Mel-5 cells...50

Figure III.8 Cephalostatin induces time dependent cell death in SK-Mel-5 cells...51

Figure III.9 Cephalostatin strongly inhibits clonogenic tumor growth ...52

Figure III.10 Caspase-dependent apoptosis in Jurkat and HeLa cells ...54

Figure III.11 Caspase-independent cell death in SK-OV-3 cells ...55

Figure III.12 Cephalostatin induced cell death in SK-Mel-5 cells is caspase-independent ....56

Figure III.13 Cephalostatin promotes STAT3 degradation ...57

Figure III.14 Cephalostatin induces G1-phase arrest in SK-Mel-5 cells...58

Figure III.15 Cephalostatin promotes survivin downregulation...59

Figure III.16 AIF is not responsible for cephalostatin induced apoptosis in SK-Mel-5 cells ...60

(7)

Figure III.18 Smac is selectively released upon cephalostatin treatment regardless of cell

type...62

Figure III.19 Cephalostatin induced Smac release is not dependent on JNK ...63

Figure III.20 Cephalostatin induced Smac release is not dependent on caspase-2 ...64

Figure III.21 Bak deficiency can not prevent Smac release and apoptosis induced by cephalostatin ...65

Figure III.22 Cephalostatin induced Smac release is partially mediated by calpain...66

Figure III.23 XIAP overexpression does not prevent cephalostatin-induced cell death ...67

Figure III.24 Smac siRNA inhibits cephalostatin-induced apoptosis ...68

Figure III.25 Caspase-9 is essential for cephalostatin-induced cell death ...69

Figure III.26 Smac silencing reduces activation of caspase-9, caspase-3 and caspase-2 but not caspase-4...70

Figure III.27 Cephalostatin activates caspase-2 ...72

Figure III.28 Caspase-2 is activated independent of caspase-9. ...73

Figure III.29 The PIDDosome is formed upon cephalostatin treatment. ...75

Figure V.1 Summary of cephalostatin-induced cell death mechanisms...84

INDEX OF TABLES

Table I.1 Anticancer agents derived from natural origin in development or clinical use (adapted from 2, 4, 5) ...2

Table I.2 Characteristics of different types of cell death (adapted from 23, 25, 26) ...8

Table II.1: summary of used cell lines ...24

Table II.2: Freezing medium...26

Table II.3 PAA-concentration in the separating gel ...35

Table II.4 Primary antibodies...38

(8)

CONTENTS VI

ABBREVIATIONS

AIF Apoptosis inducing factor

ANOVA Analysis of variance between groups Apaf-1 Apoptotic protease-activating factor-1

APS Ammonium persulfate

ASK1 Apoptosis signal-regulating kinase-1 ASK1-DN ASK-1 dominant negative

ATP/dATP Adenosine-5´-triphosphate/2´-desoxyadenosine-5´-triphosphate

Bcl B-cell lymphoma

BH Bcl-2 homology

BIR Baculoviral IAP repeats BSA Bovine serum albumin

CAD Caspase-activated DNase CARD Caspase recruitment domain CED Cell death abnormality cIAP Cellular inhibitor of apoptosis

DD Death domain

DED Death effector domain

DIABLO Direct IAP binding protein with low pI DISC Death inducing signaling complex DMSO Dimethyl sulfoxide

DNA Deoxyribonucleic acid

DR Death receptor

DTT Dithiothreitol

ECL Enhanced chemoluminescence EDTA Ethylene diamintetraacetic acid

EGTA Ethylene glycol-bis(2-aminoethylether) tetraacetic acid Endo G Endonuclease G

ER Endoplasmic reticulum

FACS Fluorescence activated cell sorter FADD Fas-associated death domain

FasL Fas ligand

FCS Fetal calf serum

FCS Forward scatter

FL Fluorescence

GI Growth inhibition

HEPES N-(2-hydroxyethyl)piperazine-N’-(2-ethanesulfonic acid) HFS Hypotonic fluorochrome solution

HtrA2 High temperature requirement protein A2 IAP Inhibitor of apoptosis protein

ICE Interleukin-1β converting enzyme IL Interleukin

(9)

IMM Inner mitochondrial membrane JNK c-Jun N-terminal kinase

kDa kilo Dalton

LB Lysogeny broth

MMP Mitochondrial membrane permeabilization

mRNA Messenger RNA

MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide NAIP Neuronal apoptosis inhibitory protein

NCI National cancer institute nt Nucleotide

OMM Outer mitochondrial membrane

PAA Polyacrylamide

PARP Poly(ADP-ribose) polymerase PBS Phosphate buffered saline PCD Programmed cell death

PI Propidium iodide

PIDD p53 inducible protein with a death domain PMSF Phenylmethylsulphonylfluoride

PS Phosphatidylserine

Q-VD-OPh N-(2-Quinolyl)valyl-aspartyl-(2,6-difluorophenoxy)methyl ketone RAIDD RIP associated ICH-1/CED-3-homologous protein with DD RNAi RNA interference

RT room temperature

SDS Sodium dodecyl sulfate

SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis SEM Standard error mean

shRNA Short hairpin RNA siRNA Short interfering RNA

Smac Second mitochondria derived activator of caspases

SSC Sideward scatter

STAT3 Signal transducer and activator of transcription 3 TBS-T Tris buffered saline with tween

TEMED N, N, N’ N' tetramethylethylene diamine TNF Tumor necrosis factor

TNF-R1 TNF receptor 1

TRAIL TNF-related apoptosis inducing ligand

UV Ultraviolet

VDAC Voltage dependent anion channel

WB Western blot

XIAP X-chromosome linked IAP

zVADfmk N-benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone

(10)

(11)

I INTRODUCTION

1 NATURAL PRODUCTS AS ANTICANCER AGENTS

Since ancient times natural products have played a major role in the treatment of diseases. The American Indians used plant extracts from Podophyllum peltatum to effectively treat skin cancers. Podophyllotoxin is the main constituent of this plant’s root and belongs to the group of anticancer agents known as podophyllins. Among this group etoposide is found, which is in regular use for the treatment of testicular teratoma and small-cell lung cancer (1).

Further discoveries based upon folk medicine were the substances vinblastine and vincristine, constituents of the plant Vinca rosea, which have significantly contributed to the successful treatment of cancer. These discoveries led to the beginning of a screening program for antitumor agents (see Table I.1) by the US National Cancer Institute (NCI) in 1960. Between 1960 and 1982 about 35,000 plant samples were tested, primarily against mouse leukemia cell lines. The most effective drug obtained from the screening was paclitaxel (Taxol), originally isolated from the bark of Taxus brevifolia (1,2).

In 1985, the NCI started a new program in which extracts from plants, animals and microorganisms (increasingly those of marine origin) were tested against a panel of 60 human cancer cell lines (1). The intention was to detect compounds that are active against solid tumors, which would have been missed in the original screen. And, in fact, almost 60% of drugs approved for cancer treatment today are of natural origin (3, 4).

The majority of chemotherapeutic drugs used in cancer therapy induce death in malignant cells through apoptosis or other kinds of programmed cell death. The inactivation of this cell death mechanism is a fundamental hallmark of cancer and leads to drug resistance. Therefore, compounds with unusual mechanisms of action are needed to circumvent chemoresistance. Natural compounds have the advantage of greater diversity than synthetic chemical libraries (5). Especially the chemical and biological diversity of the marine environment has not been acquired for a long time yet and provides great potential for the discovery of antitumor agents with novel molecular modes of action.

(12)

I INTRODUCTION

Table I.1 Anticancer agents derived from natural origin in development or clinical use (adapted from 2, 4, 5)

Plant-derived anticancer agents

Compound Source Status

Etoposide Podophyllum peltatum Phase III/IV Vinblastine, vinchristine Vinca rosea Phase III/IV Paclitaxel Taxus brevifola, Taxus baccata Phase III/IV

Microbe-derived anticancer agents

Bleomycin Streptomyces verticillus Phase III/IV Daunomycin, doxorubicin Streptomyces sp Phase III/IV Epothilone Sorangium cellulosum Phase III/IV

Marine-organism-derived anticancer agents

Bryostatin 1 Bugula neritina Phase I/II Ecteinascidin-743 Ecteinascidia turbinate Phase I/II Spongistatin 1 Hyrtios altum Preclinical

2 THE

CEPHALOSTATINS

Until the mid 1960s, investigation of natural products with marine origin was essentially non-existent. Since then, about 10,000 new structures have been isolated from marine microorganisms, sponges and e.g. marine invertebrates (1).

A promising group of compounds with marine origin comprises the family of cephalostatins (6, 7, 8, 9, 10, 11, 12, 13, 14). The cephalostatins were isolated by Prof. G. R. Pettit from the South African marine tube worm Cephalodiscus gilchristi Ridewood (family Cephalodiscidae). Until today, 19 members of this family have been characterized, all of which show the same unique growth inhibitory profile (Figure I.1) in the NCI-60 cell line screen.

(13)

Cephalostatin 1 Cephalostatin 2 Cephalostatin 1 Cephalostatin 2

Figure I.1 Fingerprint of cytotoxic profile of cephalostatin 1 and 2 evaluated by the NCI-60 screen (September 2005).

The zero value represents the mean of all cell lines tested. The bars indicate the deviation of the mean data obtained from the individual cell line from the overall mean and marks the sensitivity of the cell lines for cephalostatin 1 or 2 (negative bars, less sensitive; positive bars, more sensitive) GI50: 50% growth inhibition in the 2 days cytotoxicity assay.

(14)

I INTRODUCTION

Surprisingly, the NCI compare analysis between cephalostatin and any other compound of the NCI standard agent database shows no apparent correlation (best correlation 0.5 with teniposide), suggesting that the cephalostatins might employ unique signaling pathways, differing from any other anticancer drug. First studies carried out by our group support this notion (15, 16, 17).

Southern Northern Southern H OH cephalostatin 2 OCH3 OH cephalostatin 10 H H cephalostatin 1 R₂ R₁ H OH cephalostatin 2 OCH3 OH cephalostatin 10 H H cephalostatin 1 R₂ R₁ cephalostatin 12 Northern Unit R₂= H O N N H OH O OH OH OH H R H H O H R O O OH 1 2 O N N H OH H H H O O H O H O H

Figure I.2 Chemical structure of cephalostatin 1, 2, 10 and 12

Cephalostatin 1 and 2 are proven to be the most potent cephalostatins in the NCI-60 panel, with a GI50 value of 1 nM (see Figure I.1). In addition to the in vitro screen,

(15)

cephalostatin 1 and 2 were successfully tested in vivo on several xenografts like leukemia and melanoma.

Although the cytotoxicity profile of the cephalostatins does not differ (see Figure I.1), their potency varies depending on the chemical composition (Figure I.2). Structure-activity studies on different cephalostatins revealed that the Northern part is the most shared unit among the cephalostatins and is also strongly associated with antitumor activity (18). Cephalostatin 10 shows dose dependencies comparable to cephalo-statin 1 and 2 (6, 7, 10), whereas an increased level of hydroxylation in the Southern part results in decreased antitumor activity, as in the case of cephalostatin 12 (18).

3 AIM OF THE STUDY

Cephalostatin has been intensively studied by our research group in the last years. The suggestion based on the NCI compare analysis that cephalostatin differs from other chemotherapeutic drugs known so far was confirmed by investigating the substance in Jurkat leukemia T cells. Cephalostatin-induced cell death is not dependent on the CD95/caspase-8 pathway and does not induce the formation of the apoptosome (15), although caspase-9 is an important initiator caspase for cephalostatin in Jurkat T cells (17). Further, the induction of endoplasmic reticulum stress could be shown for cephalostatin and that caspase-4 is an initiator caspase that partially contributes to caspase-9 activation (17).

The release of different mitochondrial proteins into cytosol is a hallmark of apoptosis. Cephalostatin was found to induce an early and predominant Smac release, whereas cytochrome c could not be detected in the cytosol (15). This fact deserves attention and asks for the release mechanism and the impact of Smac on cephalostatin-mediated apoptosis.

The aim of the present work was to further investigate the underlying mechanism and characteristics of cephalostatin induced cell death based on previous work of our group (15, 16, 17), with the following main topics:

1. Is cephalostatin-induced apoptosis restricted to Jurkat T cells? 2. How is the selective Smac release mediated by cephalostatin? 3. Does Smac release influence apoptosis induction by cephalostatin?

(16)

I INTRODUCTION

4 PROGRAMMED

CELL

DEATH

The balance between cell division and cell death is of utmost importance for the development and homeostasis of a multicellular organism. Deregulation of either process has pathologic consequences and can lead to disturbed embryogenesis, neurodegenerative diseases, autoimmunity and the development of cancer. Therefore, the balance between life and death is strictly controlled and damaged or unnecessary cells are eliminated by a process called programmed cell death (PCD) (19, 20).

NECROSIS APOPTOSIS

Secondary necrosis

Phagocytosis

Figure I.3 Overview of apoptotic and necrotic cell death

Apoptosis is characterized by cell shrinkage, chromatin condensation and fragmentation of the cell in membrane enclosed apoptotic bodies. These are engulfed by macrophages (phagocytosis), thus preventing inflammation. In contrast, in necrosis the cell swells and the membrane ruptures. As a consequence cellular content is released into the surrounding tissue and inflammation is induced.

(17)

The classical ultrastructural studies of Kerr et al. (21) shed light on at least two distinct types of cell death, namely apoptosis and necrosis (Figure I.3). Apoptosis is the most common and best characterized form of PCD and was held synonymously for PCD over a long time. It is characterized by typical morphological changes such as cell shrinkage, chromatin condensation and cleavage by endonucleases and plasma membrane blebbing. Finally, the cell is fragmented into apoptotic bodies. These compact membrane-enclosed structures contain the cytosol and cell organelles. The “packaging” of the intracellular content prevents an inflammatory response, since the apoptotic bodies are engulfed by macrophages and thus removed from the tissue. An important step in this is the exposure of phosphatidylserine on the cell surface, which mediates the recognition of the apoptotic bodies by the macrophages. The morphological changes are a consequence of highly conserved, genetically controlled molecular and biochemical events, most notably mediated by caspases, a family of cysteine proteases activated in apoptosis(20).

Necrosis is – in contrast to apoptosis – an uncontrolled, passive form of death. It is usually the consequence of exposure e. g. to high concentrations of cytotoxic agents or of pathophysiological conditions, such as infection and ischemia. The swelling of the cell and rupture of the plasma membrane is a characteristic event in necrosis, resulting in the release of cellular content into the surrounding tissue and extensive inflammation. Typical attributes of apoptotic cell death like DNA fragmentation and formation of apoptotic bodies are absent in necrosis (20).

In recent years, it has become evident that the classic distinction of apoptosis and necrosis is a simplification of a highly complex system. Although caspase-mediated apoptosis is the underlying cell death program in many settings, it is unlikely that this mechanism is the only protection of an organism against unwanted and potentially harmful cells. Indeed, multiple alternative cell death pathways – even completely caspase independent ones – as well as crosstalk of PCD mechanisms are described. The various types of PCD share a common feature, namely that they are executed by active cellular processes (22, 23, 24). Since the characterization of signal transduction in alternative death styles is still in progress, one approach to classification is the nuclear morphology of the dying cell. Table I.2 gives an overview of characteristics related to different types of cell death, including morphological and biochemical features known so far.

Another approach is to classify caspase-independent cell death according to the cellular organelles involved. Thus, several models have been proposed to categorize PCD, but well defined terms are difficult to create and are probably artificial due to

(18)

I INTRODUCTION

the overlapping and commonly used signal transductions pathways between the different cell death mechanisms.

Table I.2 Characteristics of different types of cell death adapted from (23, 25, 26)

Type of cell

death Morphological changes Biochemical features Nucleus Cell

membrane Cytoplasm Apoptosis chromatin condensation,

nuclear fragmentation, DNA laddering

blebbing packaging into

apoptotic bodies caspase-dependent

Autophagy partial chromatin condensation, DNA-fragmentation very late, if at all

blebbing increased number of

autophagic vesicles caspase-independ., increased lysosomal activity

Mitotic catastrophe

multiple micronuclei, nuclear fragmentation

no consensus on the distinctive morphological appearance by now

caspase-independ. (at early stage)

Necrosis clumping and random degradation of nuclear DNA

swelling,

rupture increased vacuolation, organelle degradation, mitochondrial swelling

no energy requirement

5 APOPTOSIS SIGNAL TRANSDUCTION

Apoptosis is a tightly regulated and highly conserved cell death program which requires the interaction of multiple factors. It can be triggered by various stimuli from outside (receptor mediated) or inside (intracellular stress) the cell. Upon such a signal caspases (cysteine-dependent aspartate-specific proteases) are activated in classical apoptosis, leading to cell death.

(19)

5.1 CASPASES

Although the first caspase, interleukin-1β-converting enzyme (ICE or caspase-1), was identified in humans, the critical involvement of caspases in apoptosis was discovered in the model organism Caenorhabditis elegans. There, the gene ced-3 (cell death abnormality-3) was found to encode a cysteine protease, an essential component in developmental cell death that is closely related to the mammalian ICE. Since then, at least 14 distinct mammalian caspases have been identified, 12 of which are human. (27, 28)

5.1.1 CLASSIFICATION, STRUCTURE AND ACTIVATION

Caspases can be classified according to their structure, function and preferred substrates.

Short Prodomain CARD

INFLAMMATION DED

8,10 3,6,7,14 2,9,12 1,4,5,11

APOPTOSIS CASPASES

Figure I.4 Classification of caspases based on their prodomain structure or function

Caspases containing a long prodomain with a CARD (caspases 1, 2, 4, 5, 9, 11, 12) can be divided upon their function in inflammatory (caspases 1, 4, 5, 11) and apoptotic (caspases 2, 9, 12) caspases. Caspases-8 and 10 possess two DEDs in their long prodomain and belong to the apoptotic family members. Caspases 3, 6, 7 and 14 are apoptotic effector caspases with short prodomain.

If caspases are divided into groups by their structural characteristics, two main categories – caspases with long or short prodomain – are formed (Figure I.4).

(20)

I INTRODUCTION

Long prodomains comprise structural motifs in the death domain superfamily, including the caspase activation and recruitment domain (CARD) and the death effector domain (DED). Caspases with long prodomain (caspases 1, 2, 4, 5, 8, 9, 10, 11, 12) are enabled to interact with other proteins by these structures. This plays an important role in apoptotic signaling, since CARD and DED are the responsive elements for the recruitment of initiator caspases into death- or inflammation-inducing signaling complexes, where caspases are activated. Caspases 3, 6, 7 and 14 have short prodomains and are activated by other caspases upon proteolytic cleavage (27, 28,29).

Classification of caspases by their primary function leads to two groups comprised of inflammatory and apoptotic caspases, whereas the latter can be divided in initiator (caspases 2, 8, 9, 10, 12) and effector (caspases 3, 6, 7, 14) caspases (27).

Active site

R179 H237 C285 R341 Active caspase

Proteolytic cleavage

D119 D296

D316

Large subunit (p20) Small subunit (p10)

DED DED CARD short Pro Prodomain D119 D296 D316

Large subunit (p20) Small subunit (p10)

DED DED

CARD short Pro

Prodomain

Figure I.5 Schematic representation of proteolytic caspase activation (modified from 31)

Activation proceeds by cleavage of the N-terminal domain at Asp119, Asp296 and Asp316 (all caspase-1 numbering convention) leading to a large (p20) and a small (p10) subunit. The activity and specificity determining residues (R179, H237, C285 and R341) are brought into the necessary structural arrangement for catalysis. Cys285 is the catalytic nucleophile. The active caspase is a tetramer of two heterodimers, each comprising a large and a small subunit and an active site.

(21)

All caspases are synthesized as catalytically inactive zymogens. The zymogens consist of the different N-terminal prodomains described above followed by a large subunit of approximately 20 kDa (p20) and a small subunit of 10 kDa (p10). The subunits are separated by a small linker sequence (30) (see Figure I.5).

To become catalytically active a procaspase must undergo conformational changes and usually has to be cleaved to produce its mature form. Mature caspases are formed by association of two monomers, with each monomer comprising the large (p20) and the small (p10) subunits. Each tetramer contains two active sites formed by residues of the large and small subunit (31, 32).

Effector caspases are activated by initiator caspases through removal of the N-terminal prodomain and the linker peptide within the protease domain by cleavage at specific internal Asp residues that separate the large and small subunits. As a consequence, a conformational change occurs and the catalytic activity of the effector caspase is enhanced (31). How initiator caspases are activated is not fully understood. Two models have been proposed, namely the induced proximity and the proximity-induced dimerization model, the latter stating that the initiator caspases are recruited to large protein complexes, brought into close proximity and get activated upon dimerization (32). At present, there are different complexes described like the death inducing signaling complex (DISC), the apoptosome and the PIDDosome.

5.1.2 SUBSTRATES

Caspases recognize at least four (caspase-2 five) contiguous amino acids in their substrates P4-P3-P2-P1 and cleave after the C-terminal P1, which is usually an aspartate. The P3 position is a glutamine residue for all examined caspases, whereas the P4 position varies among different groups of caspases. Effector caspases cleave numerous proteins, including some that are responsible for structural integrity of the cell, as the DNA repair enzyme PARP (poly ADP-ribose polymerase), which is cleaved by caspase-3. Initiator caspases can activate effector caspases by proteolytic cleavage but have many other targets in the cell. For example, caspase-2 can cleave the protein golgin-160, which controls the integrity of the Golgi complex. A prominent caspase-8 substrate is the Bcl-2 family member Bid. After cleavage, Bid translocates to the mitochondria, thus promoting cytochrome c release (27). Overall, more than 280 caspase substrates are identified to date (33).

(22)

I INTRODUCTION

5.1.3 REGULATION

Because caspases play an important role in apoptosis initiation, their expression and activation state must be tightly regulated. Caspase regulation is achieved by transcriptional and posttranscriptional mechanisms. The conserved IAP (inhibitor of apoptosis) protein family can inhibit the enzymatic activity of caspases. Furthermore, caspase can be removed through proteasomal degradation promoted by IAPs. The IAP proteins were originally identified in the genome of baculovirus on the basis of their ability to suppress apoptosis in infected host cells. At least eight IAPs have been found in mammals (34, 35, 36) (see Figure I.6).

Bruce/Apollon ILP2 ML-IAP/Livin XIAP Survivin NAIP c-IAP1 c-IAP2

Figure I.6 Schematic representation of the inhibitor of apoptosis protein family (36)

IAPs have at least one baculoviral IAP repeat (BIR) domain. Additionally, most IAPs have other distinct functional domains such as the NACHT domain, the leucine-rich repeats (LRRs) and the RING (really interesting new gene) domain. The latter is an E3 ligase that presumably directs targets to the ubiquitin-proteasome degradation system. Bruce has an ubiquitin-conjugation (UBC) domain that is found in many ubiquitin-conjugating enzymes. (BIR, baculoviral IAP-repeat; cIAP, cellular IAP; IAP, inhibitor of apoptosis protein; ILP, IAP-like protein; ML-IAP, melanoma IAP; NAIP, neuronal apoptosis-inhibitory protein; NACHT, domain found in NAIP; XIAP, X-chromosome-linked IAP.)

The hallmark of IAPs is the baculoviral IAP repeat (BIR) domain, a ~80 amino acid zinc binding domain. XIAP, the most extensively studied IAP member contains three BIR domains with different functions: BIR3 potently inhibits the activity of processed caspase-9, whereas the linker region between BIR1 and BIR2 targets caspase-3 and -7. The IAP mediated inhibition of caspases is antagonized by a family of proteins

(23)

that contain an IAP-binding motif like the mitochondrial protein Smac/DIABLO (see chapter I5.3.1) and Omi/HtrA2. Except for survivin, all other IAPs contain other functional domains such as a RING domain, an E3 ligase that presumably directs targets to the ubiquitin proteasome degradation system (34, 35).

Survivin, the smallest member of the IAP family with a single BIR domain, is transcriptionally regulated by the oncogenic transcription factor STAT3 (37). The BIR domain of survivin is closely related to the BIR3 of XIAP and several studies showed that survivin is capable of binding caspases -3, -7 and -9 but this is still controversially discussed (38, 39). Besides its function in controlling apoptosis, the IAP member survivin seems to regulate the mitotic progression and the production of the survivin protein is upregulated in G2/M phase.

5.2 EXTRINSIC

PATHWAY

There are two alternative pathways – the extrinsic and the intrinsic pathway - that initiate apoptosis depending on the origin of the death stimuli. The extrinsic pathway is mediated by death receptors on the cell surface and has a crucial role during development and for the immune system. The death receptors (DR) belong to the tumor necrosis factor (TNF) receptor superfamily, characterized by an extracellular ligand binding domain and an intracellular death domain. In mammals several death receptors are known as TNF-R1, TRAIL or CD95 (= Fas, APO-1), the latter being the most extensively studied. The TNF receptor superfamily includes the decoy receptors, which lack a functional death domain, thus building a negative regulation mechanism. (19, 40)

The extrinsic pathway (Figure I.7) is initiated upon binding of an extracellular ligand such as CD95L to its receptor (in this case CD95). After ligation, micro-aggregates are formed on the cell surface leading to the attraction of the intracellular adaptor protein FADD (Fas-associated death domain protein) via the death domains. FADD, in turn recruits the inactive caspase-8 or -10 zymogens through interaction with their death effector domain (DED) to the so called DISC (death inducing signaling complex). At the DISC the initiator caspases get active, which is in type I cells sufficient to initiate apoptosis directly via the induction of downstream effector caspases like caspase-3 and -7. (19)

In type II cells the amount of active caspase-8 is not enough, so an amplification of the apoptotic signal via the mitochondria takes place. This crosstalk is mediated by

(24)

I INTRODUCTION

the cleavage of Bid into truncated Bid (tBid) by active caspase-8. Subsequently tBid translocates to the mitochondria and induces the release of apoptotic proteins like cytochrome c (41). DD DD DE D DE D

Effector caspases

tBid

DISC

Proc

as

pa

se

-8

death receptor (e.g. CD95)

CD95L

active

caspase-8

FADD

Figure I.7 The extrinsic apoptotic pathway

On binding of CD95 ligand to its receptor CD95, trimerization takes place and FADD is recruited to the cytosolic domain via interaction with the death domain (DD). Caspase-8 binds to the complex thus forming the death inducing signaling complex (DISC), where it is activated. An amplification of the apoptotic signal is possible upon caspase-8 mediated cleavage of Bid, which translocates to mitochondria.

(25)

5.3 INTRINSIC

PATHWAY

Mitochondria, the main energy producers of the cell, are essential for maintaining cellular life and play a major role in the apoptotic process at the same time. Besides amplifying the extrinsic apoptotic pathway, mitochondria are the central organelles involved in the propagation of death signals originating from inside the cell. Such signals can be for example DNA-damage, oxidative stress or signals induced by chemotherapeutic drugs. The central event of intrinsic apoptosis induction is the mitochondrial membrane permeabilization (MMP) of the outer (OMM) and the inner (IMM) mitochondrial membrane. MMP causes the dissipation of the mitochondrial membrane potential (∆ψm), which is required for mitochondrial functions as ion

transport or energy conservation. When outer membrane integrity is lost proapoptotic proteins from the mitochondrial inter-membrane space are released into cytosol and either activate caspases or act in a caspase-independent manner leading to cell death. Cytochrome c, Smac/Diablo (second mitochondria derived activator of caspases / direct IAP binding protein with low pI), Omi/HtrA2 (high temperature requirement protein A2), AIF (apoptosis inducing factor) and EndoG (endonuclease G) belong to these apoptotic factors (42, 43).

AIF translocates from mitochondria into the nucleus and causes caspase-independent cell death. It induces chromatin condensation and high molecular weight DNA fragmentation. AIF is a flavoprotein with dual roles in life and death since besides its role in apoptosis its participation in the formation of the respiratory complex I was shown. EndoG also translocates to the nucleus and mediates internucleosomal DNA-fragmentation (44).

Cytochrome c is involved in electron transport and induces upon apoptotic stimuli the energy-dependent (ATP/dATP) formation of the apoptosome (Figure I.8). This oligomeric complex consists of the protein Apaf-1 (apoptotic protease activating factor 1) whose conformation is changed after binding to cytochrome c, ATP/dATP and procaspase-9. In the apoptosome complex caspase-9 is activated and initiates the caspase cascade, finally leading to cell death (19, 45).

For the prevention of accidental caspase activation there is a negative regulatory mechanism by the inhibitors of apoptosis proteins (IAPs) (see I.5.1.3). These in turn are inactivated by Smac (see I.5.3.1) and Omi/HtrA2. Omi is a serine protease that has been reported to cleave cIAPs and to participate in caspase-independent apoptosis (40, 46, 47, 48).

(26)

I INTRODUCTION

Apoptotic stimulus

MITOCHONDRIA

Cyt c Procaspase-9 CARD Apaf-1 ATP Active caspase-9 Effector caspases Smac IAPs IAPs CARD Bcl-2 Bcl-2 Bak Bak Bid, Bax AIF,EndoG, Omi

Apoptosome

CASPASE-INDEPENDENT CELL DEATH

Figure I.8 The intrinsic apoptotic pathway

Mitochondria are the central organelles in the intrinsic apoptosis pathway. Upon many different stimuli like chemotherapeutic drugs, the mitochondria membrane is permeabilized and proapoptotic proteins are released into cytosol. A complex is formed consisting of cytochrome c, Apaf-1 and procaspase-9, where this caspase is activated and initiates the activation of effector caspases. Smac abolishes the negative regulation on caspases by IAP. AIF, Omi and EndoG are supposed to induce caspase-independent cell death. Bcl-2 family proteins (e.g. Bax, Bid) regulate the intrinsic pathway.

(27)

5.3.1 SMAC

Smac is the best known antagonist of the IAP family and reactivates processed initiator as well as effector caspases through distinct mechanisms, although both require physical interaction with IAPs. Smac is a constitutively expressed protein located in inter-membrane space. Its N-terminal 55 residues encode a mitochondria-targeting sequence, which is proteolytically removed to generate the 23 kDa mature Smac on entry into the mitochondria. This cleavage results in the exposure of four hydrophobic amino acids (Ala-Val-Pro-Ile), the tetrapeptide IAP binding motif at the N-terminus (30).

Caspase-9 contains a similar internal IAP binding tetrapeptide motif (Ala-Thr-Pro-Phe), which is exposed after activation (proteolytic cleavage). This leads to the recruitment of XIAP to active caspase-9, thus inhibiting the caspase. Smac binds via its IAP binding motif to a highly conserved surface groove on the BIR3 domain of XIAP and competitively displaces the bound caspase-9, thus reactivating it (Figure I.9). IAP smac IAP IAP IAP active caspase inhibited caspase smac

Figure I.9 Simplified illustration of removal of IAP-mediated caspase-inhibition by Smac

Smac can bind various IAPs through its N-terminal tetrapeptide binding motif. In case of initiator caspase-9, which has a similar IAP binding motif, active caspase-9 is displaced competitively with Smac from XIAP. The inhibition of effector caspases by XIAP is abolished by sterical reasons after binding of Smac to BIR2 of IAPs.

Smac plays a less immediate role in removing the IAP-mediated inhibition of effector caspases, since the binding site of the IAP binding motif of Smac maps to BIR2 and BIR3, but the IAP fragment responsible for inhibiting caspase-3 and -7 is the linker between the BIR1 and BIR2 domain of XIAP. Although the mechanism is not fully

(28)

I INTRODUCTION

understood, modeling studies suggest, that steric clashes preclude the simultaneous binding of BIR2 to effector caspases, once Smac has bound to it (30, 34, 49).

5.3.2 APOPTOSIS REGULATION BY THE BCL-2 PROTEIN FAMILY

The Bcl-2 (B cell lymphoma) family is an evolutionary conserved group of proteins, critically involved in the regulation of the release of apoptogenic factors from mitochondria in the intrinsic apoptosis pathway. On the basis of functional and structural criteria the Bcl-2 proteins can be divided in three groups (Figure I.10). The first group contains antiapoptotic proteins characterized by four Bcl-2 homology (BH) domains, called BH1 – BH4. Most of these proteins possess a hydrophobic tail, which allows the attachment to membranes of organelles like mitochondria. Bcl-2 and Bcl-xL as representatives of this group prevent the release of apoptogenic factors

from mitochondria and therefore protect against outer membrane permeabilization by a not fully understood mechanism (50, 51).

BH3 BH1 BH2 BH4 Transmembrane Group I Group II Group III e.g. Bcl-2 e.g. Bax e.g. Bid

e.g. Bik BH3-only

Figure I.10 Bcl-2 family members (modified from 40)

On structural and functional basis Bcl-2 proteins are divided into three groups. Group I is characterized by 4 BH (Bcl-2 homology) domains, a membrane anchoring tail (Transmembrane) and antiapoptotic function. Group II consists of proapoptotic members like Bax and Bak. The third group has just one BH domain and regulates the function of group I and II.

Group II includes e.g. Bax and Bak, proapoptotic members which are structurally similar to group I but lacking the BH4 domain. Bax is loosely attached to the outer membrane or sequestered in cytosol, whereas Bak has an anchor that attaches it to the mitochondrial outer membrane in a complex with the VDAC (voltage-dependent

(29)

anion channel). Upon a death stimulus Bak and Bax undergo a conformational change, oligomerize and induce the formation of a pore in the OMM through which e.g. cytochrome c and Smac are released. Another model proposes that Bax targets one or more components of the permeability transition in the IMM (40, 52).

Group III, also known as BH3-only proteins, has many members whose common feature is the presence of a single BH3 domain. These proteins fulfill their proapoptotic function either by activating proapoptotic proteins like Bax or by inhibiting antiapoptotic Bcl-2 proteins. Bid for example is thought to induce the conformational change in Bax/Bak, which leads to insertion in the OMM.

5.4 PIDDosome

Recruitment to large macromolecular complexes is a critical step in activating initiator caspases. This is well established for caspase-8, which is recruited to the death inducing signaling complex (extrinsic pathway, see I.5.2) and for caspase-9, which is activated in the apoptosome (intrinsic pathway, see I.5.3) complex. Besides these two complexes the inflammasome is known with the inflammatory initiator caspases -1 and -5 as constitituents (53).

Caspase-2 is one of the most conserved caspases and shares the commonness of the initiator caspases named above: it contains a CARD, the responsible structural element for recruitment to the described protein complexes.

CARD CARD DD DD

Procaspase-2 RAIDD PIDD

LRR Interaction Interaction

Figure I.11 The PIDDosome

Caspase-2 can be activated via the PIDDosome. This complex consists of procaspase-2, the adaptor protein RAIDD and the p53-inducible protein with a death domain (PIDD). Procaspase-2 and RAIDD can interact via their caspase-recruitment domain (CARD). RAIDD interacts with PIDD via a death domain (DD). PIDD also contains LRR (leucine rich repeats), a protein interaction motif found in various proteins with diverse function.

(30)

I INTRODUCTION

Indeed, a complex for the activation of caspase-2 was recently described as well. The so called PIDDosome (Figure I.11) is a large protein complex with a molecular weight in excess of 670 kDa. It consists of the adaptor protein RAIDD (RIP-associated ICH1/CED3-homologous protein with a death domain) and PIDD (p53-induced protein with a death domain). RAIDD contains a CARD through which it can bind caspase-2 and a DD that allows interaction with PIDD (54, 55, 56).

Besides the death domain PIDD contains N-terminal leucine-rich repeats (LRRs), protein interaction motifs probably for recognizing signals of unknown nature (53, 56, 57).

Activation of the PIDDosome is described to sensitize cells to genotoxic stress, but in addition to DNA-damage cell lysis under hypotonic conditions initiates the assembly (56).

At present, it is unclear whether additional factors are involved in the assembly of the PIDDosome.

5.5 APOPTOSIS DEREGULATION – TARGETS FOR DRUG

DISCOVERY

Deregulation of apoptosis can disrupt the balance between proliferation and cell death and can lead to diseases as cancer. In many cancers, proapoptotic proteins have inactivating mutations or are deleted or the expression of antiapoptotic proteins is upregulated. Cancers that possess alterations in proteins involved in the extrinsic or intrinsic cell death signaling are often resistant to chemotherapy. The metastatic melanoma for instance still has poor prognosis with response rates ranging from 10 to 25% and a mean survival of 8 month for single dacarbazine treatment. Neither combination of dacarbazine with other drugs nor immunotherapy could effectively improve the therapy outcome. Therefore, new strategies to restore programmed cell death are needed and might be effective against many cancers. One promising approach is the investigation of the altered signal network in cancer growth regulation in order to act with specificity at molecular level. At present, agonistic antibodies against TRAIL receptors or antisense oligonucleotides targeting the antiapoptotic protein Bcl-2 are in clinical trials. Inhibitors of the antiapoptotic IAP family are under preclinical examination (58, 59, 60).

In this respect STAT3 (signal transducer and activator of transcription) is – among many others – a very interesting therapeutic target, since it joins numerous

(31)

oncogenic signaling pathways and is constitutively activated at 50 – 90% frequency in diverse human cancers, whereas it is tightly controlled and transiently activated in non-transformed cells (61). Persistent STAT3 activity leads to profound changes in gene expression patterns altering cell survival and proliferation, angiogenesis and metastasis and immune evasion (61).

persistent activity ST A T 3 P_P Survival: ↑Bcl-x_L ↑Mcl-1 ↑survivin ↓p53 Proliferation: ↑Myc ↑Cyclin D1/D2 ↓p53

Figure I.12 Consequences of persistent STAT3 activity

Constitutive activity of STAT3 leads to alterations in the regulation of cell survival and proliferation. ↑ = upregulation; ↓ = downregulation.

The first anti-apoptotic factor found to be regulated by STAT3 was Bcl-xL, but since

then many other proteins, e.g. survivin or cyclin D1, that are crucial for tumor cell proliferation and survival have been found to be controlled by this transcription factor (Figure I.12). First studies have shown that interruption of survivin activity via phosphorylation mutants or inhibition of protein synthesis via antisense oligonucleotides leads to caspase-dependent and caspase-independent PCD (62, 63, 64).

Survivin is ubiquitously expressed during development but is absent in most adult tissues. In contrast, it is highly expressed in cancer cells and associated with decreased patient survival, making survivin as well as STAT3 attractive diagnostic and therapeutic targets (64).

(32)

II MATERIALS AND METHODS

1 MATERIALS

1.1 CEPHALOSTATIN

Cephalostatin 2, cephalostatin 10 and cephalostatin 12 were isolated as described in (6, 11, 10). The substances were kindly provided by Prof. G. R. Pettit (Cancer Research Institute, Arizona State University, Tempe, USA). The 10 mM stock solutions prepared in DMSO were stored at -20°C.

1.2 REAGENTS

Reagent Company

Complete Roche, Mannheim,

Germany

DMEM PAN Biotech, Aidenbach, Germany

DMSO Roth GmbH, Karlsruhe,

Germany

Etoposide Calbiochem, Schwalbach, Germany

FCS gold PAN Biotech, Aidenbach, Germany

G418 sulfate PAA Laboratories, Cölbe, Germany

Hoechst 33342 Sigma, Taufkirchen, Germany

McCoy´s 5a PAN Biotech, Aidenbach, Germany

Mitotracker Red Molecular Probes, Karlsruhe, Germany MTT Sigma, Taufkirchen,

Germany

Paclitaxel Sigma, Taufkirchen, Germany

Reagent Company

Polyacrylamide Roth GmbH, Karlsruhe, Germany

Propidium iodide Sigma, Taufkirchen, Germany

Puromycin PAA Laboratories, Cölbe, Germany

Q-VD-OPh Calbiochem, Schwalbach, Germany RPMI 1640 PAN Biotech,

Aidenbach, Germany SP600125 Calbiochem,

Schwalbach, Germany Staurosporine Calbiochem,

Schwalbach, Germany Thapsigargin Sigma, Taufkirchen,

Germany

zVAD-fmk Calbiochem, Schwalbach, Germany zVDVAD MBL, Woburn, USA

(33)

2 CELL

CULTURE

2.1 CELL

LINES

2.1.1 HUMAN LEUKEMIA JURKAT T CELL LINES

All human leukemia Jurkat T cell lines were cultivated in RPMI 1640 containing 2 mM L-glutamine supplemented with 10% fetal calf serum (FCS) and 1% pyruvate.

Caspase-9-deficient and -reconstituted Jurkat T cells (65) and Bak-deficient and reconstituted Jurkat T cells (courtesy of Prof. Dr. K. Schulze-Osthoff, Düsseldorf, Germany) were cultured in the medium described above containing heat-inactivated FCS.

Jurkat cells stably overexpressing XIAP were provided by Dr. C. Duckett (University of Michigan). These cells were cultivated in the medium described for Jurkat T cells in the presence of 1 µg/ml puromycin every fifth passage.

2.1.2 CARCINOMA CELL LINES

The human cervix carcinoma cell line HeLa was obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany). The human melanoma cell line SK-Mel-5 was obtained from the American Type Culture Collection (ATCC, Manassas, USA). HeLa and SK-Mel-5 cells were cultivated in DMEM containing 2 mM L-glutamine supplemented with 10% fetal calf serum and 1% pyruvate. The human ovarian cancer cell line SK-OV-3 was obtained from ATCC and cultivated in McCoy´s 5a supplemented with 10% fetal calf serum. MCF-7 cells and caspase-3 reconstituted MCF-7 cells were kindly provided by Prof. Dr. K. Schulze-Osthoff. MCF-7 stably transfected with either SmaYFP or cytochrome c-GFP (66) were provided by Prof. Dr. J.H.M. Prehn. All MCF-7 cells were grown in RPMI 1640 containing 2 mM L-glutamine supplemented with 10% heat-inactivated fetal calf serum. See Table II.1 for a summary of the used cell lines.

(34)

Table II.1: summary of used cell lines

Jurkat T cell clones Carcinoma cell lines

Wild type J16 HeLa

Caspase-9-deficient cells SK-Mel-5 Caspase-9-reconstituted cells SK-OV-3

XIAP-overexpressing Jurkat cells MCF-7 +/- Caspase-3

Bak-deficient cells MCF-7 smac-YFP

Bak-reconstituted cells MCF-7 cytochrome c-GFP

2.2 CULTIVATION

All cell lines were cultured at 37°C and 5% CO2 in humified atmosphere. Cell density

and viability was determined by staining with trypan blue using a VI-CELLTM_cell

viability analyzer (Beckman Coulter, Krefeld, Germany).

The Jurkat subclones were split three times a week and diluted with prewarmed medium to 1 x 105cells/ml respectively 7 x 104 cells/ml before weekends. Cell density never exceeded 1 x 106 cells/ml in order to maintain genetic stability. For the same reason cells were not used for experiments any longer after reaching passage 20. HeLa, SK-Mel-5, SK-OV-3 and MCF-7 cells were grown as monolayer and split when reaching 80 - 90% confluence. Briefly, cells were washed with prewarmed PBS (see below) and detached with 3 ml Trypsin/EDTA (see below) / 75 cm2 flask. After detaching, Trypsin/EDTA was inactivated by adding 7 ml medium and cells were centrifuged (180 x g, 10 min, RT). Cells were resuspended in fresh medium and 1 - 3 x 106_{cells were transferred to 75 cm}2_{cell culture flasks.}

PBS (pH 7.4) NaCl 7.2 g Na2HPO4 1.48 g KH2PO4 0.43 g H2O ad 1,000 ml Trypsin/EDTA (T/E) Trypsin 0.50 g EDTA 0.20 g PBS ad 1,000 ml

(35)

2.3 SEEDING FOR EXPERIMENTS

Jurkat leukemia T cells were seeded approximately 16 h before experiments at a density of 0.5 x 106 _{cells/ml. Alternatively, cells were seeded 2-3 h before}

stimulation, with higher density (0.7 x 106 cells/ml). Cells were centrifuged (180 x g, 10 min, RT), resuspended in prewarmed medium and cell density was determined by VI-CELLTM cell viability analyzer. Cells were diluted with medium to the desired concentration and seeded in 24-well tissue culture plates for all experiments except MTT viability assay (96-well plates, see II .4) and clonal survival assay (6-well plates, see II .6).

Carcinoma cell lines were detached and centrifuged as described in II2.1.2. Cell concentration was adjusted to 0.2-0.3 x 106 cells/ml and seeded in 12- or 24-well plates the day before the experiment. Before stimulating the cells medium was removed and replaced by fresh medium.

2.4 FREEZING AND THAWING

In order to preserve a sufficient stock of each cell line, long term storage took place in liquid nitrogen. Therefore, cells in low passages were frozen in special medium (Table II.2: Freezing medium), containing DMSO for avoiding cell rupture. Cells were centrifuged (180 x g, 10 min, 4°C) and resuspended in ice-cold freezing medium at a concentration of 2-3 x 106 cells/ml. 1.5 ml cell suspension was transferred into each cryovial and frozen at -20°C over night. Afterwards, vials were stored at -80°C for permanent usage or transferred into liquid nitrogen after two days for long term storage.

Cells were defrosted by gently dissolving in 10 ml prewarmed medium. Subsequent, cells were centrifuged (180 x g, 10 min, RT) to remove DMSO and dead cells. After resuspension in fresh medium, cells were left to grow for at least five days before any experiments.

(36)

Table II.2: Freezing medium

HeLa Jurkat MCF-7 SK-Mel-5 SK-OV-3

RPMI 1640 - 70% 70% - - DMEM 80% - - 80% - McCoy´s 5a - - - - 85% FCS gold 10% 20% 20% 10% 10% DMSO 10% 10% 10% 10% 5%

3 FLOW

CYTOMETRY

Flow cytometry is a technology that allows the measurement of multiple physical characteristics of single particles at the same time. These particles, usually cells, are suspended in a stream of fluid and pass through a beam of light. The list of measurable parameters includes properties like a particle’s relative size, relative granularity or internal complexity and relative fluorescence intensity. Among other things, this method is suitable for measuring cell cycle, apoptosis, viability, protein expression and localization, cell surface antigens and enzymatic activity.

(37)

The fluid stream transporting the cells should be positioned in the center of the laser beam to yield optimal illumination and the cells or particles should move through the laser beam one by one. For this reason the sample is injected into a stream of sheath fluid within the flow chamber, where the sheath fluid accelerates the particles and restricts them to the center of the sample core (see Figure II.1). This process is called hydrodynamic focusing.

When particles pass the laser beam, the light is scattered and simultaneously, if the particles have been stained with fluorescence dyes able to absorb the laser light, fluorescence occurs. The scattered and fluorescent light is collected by lenses and forwarded to the appropriate detectors.

Morphological parameters like the relative size and granularity of a cell influences the light scattering. In line with the laser beam the forward scatter (=FSC) proportional to cell size is measured. Light scattered perpendicular to the laser is called sideward scatter (SSC) and is characteristic for the internal complexity. Fluorescence was measured by using the appropriate filters for the respective fluorochromes (e.g. FL2 for detection of propidium iodide). All measurements were performed on a FACSCalibur (Becton Dickinson, Heidelberg, Germany), equipped with a 488 nm argon laser. Sheath fluid is composed as seen below (FACS buffer). FACS buffer (pH 7.37) NaCl 8.12 g KH2PO4 0.26 g Na2HPO4 2.35 g KCl 0.28 g Na2EDTA 0.36 g LiCl 0.43 g Na-azide 0.20 g H2O ad 1,000 ml

3.1 NICOLETTI

ASSAY

A simple and rapid method for quantification of apoptosis is the measurement of DNA fragmentation, a characteristic event caused by the activation of endonucleases. According to the method of Nicoletti et al. (68) cells are permeabilized in a hypotonic buffer (HFS buffer, hypotonic fluorochrome solution),

(38)

which contains propidium iodide for staining the DNA. The resulting red fluorescence is measured by flow cytometry. Figure II.2 shows a characteristic histogram of untreated control cells after staining with propidium iodide. Fluorescence intensity is proportional to DNA content resulting in a peak containing 2n DNA content (G0/G1), whereas cells in G2/M phase emit higher

fluorescence due to 4n DNA content. Upon eluting the low molecular weight fragments of apoptotic cells by the hypotonic buffer less dye is taken up and the resulting hypodiploid region left to the G0/G1 peak is considered apoptotic (sub

G1). G1 sub G1 G2 S G1 sub G1 G2 S

Figure II.2 Histogram of untreated PI-stained cells

Jurkat cells were permeabilized and stained with propidium iodide (PI). A typical histogram of untreated cells is shown with different peaks representing cell cycle distribution. The region left to the G1 peak is considered apoptotic (sub G1).

Protocol: Jurkat cells and adherent cell lines were seeded as described in chapter II2.3 and either left untreated or stimulated with the required substances. After different incubation times cells were harvested by centrifugation (600 x g, 10 min, 4°C) and washed once with cold PBS. Cells were incubated in 250 µl (Jurkat cells) or 500 µl (adherent cell lines) HFS buffer (see below) overnight at 4°C and analyzed by flow cytometry. The percentage of sub G1 region was determined as a

parameter of apoptotic cells.

HFS buffer

Sodium citrate 0.1% (w/v) Triton X-100 0.1% (v/v) PBS ad 1,000 ml

(39)

3.2 PHOSPHATIDYLSERINE

TRANSLOCATION

An early event in apoptosis is the loss of cell membrane asymmetry. In healthy cells, the phospholipid phosphatidylserine (PS) is located on the cytoplasmic side of the membrane. Upon apoptotic stimuli it translocates to the outer leaflet of the cell membrane. The annexin V assay makes use of this exposure of PS for the detection of cells in early or immediate apoptosis, even before events like DNA fragmentation can be measured. Annexin V is a small Ca2+_{dependent protein with}

a high and selective affinity for PS. It can be tagged with FITC without compromising its binding properties to PS thus enabling the marked cells to be analyzed by flow cytometry. An additional staining with PI opens the opportunity to distinguish between live cells (Annexin V negative, PI negative), apoptotic cells (Annexin V positive, PI negative) and necrotic cells (Annexin V positive, PI positive).

Protocol: Phosphatidylserine translocation was analyzed by the Annexin V-FITC Detection Kit (BenderMed Systems, Vienna, Austria) according to the manufacturer’s instructions. Briefly, cells were either left untreated or stimulated with the required substances for different periods of time and collected by centrifugation (600 x g, 4°C, 10 min). They were washed once with cold PBS, resuspended in 1x binding buffer and incubated with Annexin V-FITC solution for 15 min at room temperature. After another centrifugation step (600 x g, 4°C, 10 min), the pellet was resuspended and PI solution was added. The probes were immediately analyzed by FACS. Only Annexin V positive and PI negative cells were considered apoptotic.

4 MTT VIABILITY ASSAY

The mitochondrial respiratory activity is a parameter for cell viability. Though, a method for cytotoxicity determination of a substance is to measure this activity by the MTT assay. This colorimetric assay uses the tetrazolium salt MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide). It is based on the reduction of MTT by enzymes of the mitochondrial electron transport assembly, leading to the formation of a blue formazan derivative. The reduction is

(40)

proportional to the activity of the assembly and thus to the vitality of the cells. The absorption of the formazans can be measured at 550 nm in a spectrophotometer. Protocol: Cells were seeded at a concentration of 7 x 105_{(Jurkat cells) or}

0.15 x 105_{(SK-Mel-5) cells/ml in a 96-well plate (100 µl per well) the day before}

stimulation. After stimulation 10 µl of MTT solution (stock solution: 5 mg/ml in PBS, sterile filtered and kept in aliquots at –20°C) was added to each well and incubated at 37°C for 60 minutes. Afterwards, cells were lysed by adding 190 µl DMSO to each well and shaking the plates in the dark for another hour. Finally, the absorption of the solubilized formazan crystals was measured at 550 nm in an ELISA plate reader (SLT spectra, SLT Labinstruments, Crailsheim, Germany).

5 MICROSCOPY

5.1 LIGHT

MICROSCOPY

The characteristic morphological changes of apoptosis as well as other forms of programmed cell death, such as shrinking, swelling or formation of apoptotic bodies can be easily detected by light microscopy.

Cells were left untreated or stimulated with the required substances for different periods of time. Cells were viewed with a Zeiss Axiovert 25 microscope (Zeiss, Oberkochen, Germany) at 40 x magnification and images were obtained with a connected reflex camera.

5.2 FLUORESCENCE MICROSCOPY

A characteristic feature of an apoptotic cell is condensation of DNA followed by its fragmentation. Vital staining of DNA with Hoechst 33342 allows visualization of DNA changes in a fluorescence microscope. The dye is cell permeable and intercalates in the DNA due to its planar structure. Healthy cells emit only weak blue fluorescence since the DNA is distributed evenly in the nucleus. The nucleus of apoptotic cells is smaller in size and due to the condensed DNA shows a strong blue signal.

(41)

Protocol: Cells were either left untreated or stimulated with cephalostatin for various periods of time. 10 µl of Hoechst solution (0.1 mg/ml in H2O) were added

to each well and the plate was incubated at 37°C for approximately 5 min. Subsequently, pictures were taken with a Zeiss Axiovert 25 microscope (Zeiss, Jena, Germany) and connected camera.

5.3 CONFOCAL

MICROSCOPY

In a conventional light microscope all object points are illuminated parallel, whereas the organism in a confocal LSM is irradiated in a point wise manner. The out of focus information is eliminated by a pinhole, allowing high-quality images with a maximum resolution. Confocal imaging enables three-dimensional studies of thick specimens and colocalization of signals from different fluorochromes. Protocol: for the visualization of Smac and cytochrome c release a LSM 510 Meta (Zeiss, Oberkochen, Germany) was used. MCF-7 cells stably expressing either cytochrome c-GFP or Smac-YFP (66) were seeded on glass coverslips in 24-well plates and grown overnight. Cells were stimulated with 1 µM cephalostatin or 2 µM staurosporine. 1 hour prior to the end of stimulation cells were stained with 100 nM Red 580 (Molecular Probes, Karlsruhe, Germany). Cells were washed 3 times with PBS and fixed with 3% paraformaldehyde in PBS for 15 min at room temperature and washed again three times with PBS. Glass coverslips were then covered with a droplet of fluorescent mounting medium and mounted on a microscope slide.

6 CLONOGENIC

ASSAY

Clonogenic assay or colony formation assay is an in vitro long term cell survival assay to determine the effectiveness of cytotoxic agents. It is based on the ability of a single cell to grow into a colony. The assay essentially tests every cell in the population for its ability to undergo “unlimited” division.

Protocol: SK-Mel-5 cells were seeded as described in II.2.3 and treated with 50 nM cephalostatin or 50 nM taxol for 2 hours. Afterwards cells were detached and washed with PBS to remove eventually remaining substances. Cell count was

(42)

determined and 1 x 104 SK-Mel-5 cells were seeded in 6-well plates. Cells were left to grow for 8 days and colonies were stained with crystal violet. Since SK-Mel-5 cells don’t posses the ability to form colonies, but grow as loosely spreaded network, instead of counting the colonies crystal violet was solved and absorption at 550 nm was measured. Untreated control cells were set 100% viability.

7 WESTERN

BLOT

Western blot is a method to detect specific proteins present in a given sample, for example a cell lysate. Denaturized proteins are first separated by mass using gel electrophoresis and then transferred onto a membrane. Afterwards proteins can be visualized by immunodetection using a specific antibody.

7.1 SAMPLE

PREPARATION

7.1.1 WHOLE CELL LYSATES

General lysis buffer

Tris-HCl, pH 7.5 30 mM NaCl 150 mM EDTA 2 mM Triton X-100 1% CompleteTM Sample buffer (5x) 3.125 M Tris-HCl, pH 6.8 100 µl Glycerol 500 µl SDS 20% 250 µl DTT 16% 125 µl Pyronin Y 5% 5 µl

Lysis buffer for phosphorylated proteins Tris-HCl, pH 7.5 20 mM NaCl 137 mM Na4P2O7 2 mM EDTA 2 mM C3H7Na2O6P (Na glycerolphosphate) 20 mM NaF 10 mM Na3VO4 2 mM PMSF 1 mM Triton X-100 1% Glycerol 10% CompleteTM H2O ad 1 ml

(43)

Protocol: Jurkat cells or adherent cell lines were seeded (see II.2.3) and left untreated or stimulated with cephalostatin 2 or the respective positive controls. After incubation cells were harvested by centrifugation (1500 rpm, 10 min, 4°C) and washed once with cold PBS. The adherent cell lines had to be detached by Trypsin/EDTA and were also centrifuged and washed. Pellets were resuspended in the appropriate lysis buffer (100 µl for three wells) and incubated on ice for 30 min or stored at -20°C. Subsequently, lysates were centrifuged at 10,000 x g, 4°C for 10 min. Supernatants were transferred to new tubes and protein concentration was determined by the Bradford method as described in IV.7.2. Lysates were diluted 1:5 with 5 x sample buffer and boiled at 95°C for 5 min. Afterwards, samples were stored at -20°C or used immediately for western blot analysis. PMSF, Na3VO4 and Complete™ were added to the lysis buffers immediately

before use.

7.1.2 CYTOSOLIC AND MITOCHONDRIA CONTAINING FRACTIONS

In apoptosis, molecules like cytochrome c and Smac normally localized in the intermembrane space of mitochondria are released into the cytosol. There they are part of activation complexes for caspases or they translocate into the nucleus where they participate in DNA fragmentation (Endonuclease G, AIF). For analyzing the release of these factors, the cytosol has to be separated from the mitochondria. This is accomplished by a permeabilization buffer containing a low concentration of digitonin which forms complexes with cholesterol in the cell membrane. Thus, small pores develop through which the cytosol is eluted into the iso-osmotic buffer while organelles are retained inside the cell. The protocol was carried out as described previously (15).

Permeabilization buffer (pH 7.2) Mannitol 210 mM Sucrose 70 mM Hepes pH 7.2 10 mM EGTA 0.2 mM Succinate 5 mM BSA 0.15% (w/v) Digitonin 60 µg/ml

Unusual apoptotic signaling pathways in cancer cells induced by cephalostatin